This project is using a combination of methods to analyze the ion transport properties of lysosomal membranes. Lysosomes are intracellular organelles that serve in most cells as digestive organelles although in some tissues they are used for other functions. Disorders of lysosome function lead to a variety of diseases including neurological dysfunction (lysosomal storage diseases) and osteopetrosis (overcalcification of bone). Lysosomes utilize an ATP-driven proton pump to maintain an acidic luminal pH and facilitate their digestive function. Such a pump can only be effective if accompanied by additional ion transport to dissipate the transmembrane voltage built up by the ATPase, a counterion pathway. We recently used isolated lysosomes to identify and characterize a Chloride permeability in the lysosomal membrane which has the features required of such a counterion pathway and demonstrated that the chloride is transported by ClC-7, a Cl-/H+ antiporter specifically targeted to the lysosomal membrane. Over the past fewyears we have been working to develop methods to accurately meaure the pH in lysosomes in living cells in order to determine the influence of ClC-7 and other transporters on the lysosomal pH. These methods use dual-wavelength ratiometric fluorophores linked to dextran to specifically target lysosomes. pH is measured by processing images of the cells taken at the two wavelengths. We have been collaborating with investigators in CIT to develop algorithms to accurately reconstruct 3-Dimensional maps of all dextran-labeled lysosomes in a cell; these methods will be useful for understanding the pH distribution among lysosomes. Last year we have worked out methods to accurately calibrate the system to correlate image measurements with actual pH values. We showed that the siRNAs used to knock down ClC-7 expression affect pH not only in the knockdown cells, but in control cells as well, we are currently seeking alternative methods to manipulate ClC-7 levels in living cells. Also in the past year we have worked out fluorescence-based assays to measure the membrane voltage in isolated lysosomes. This is a useful to to analyze the relative contributions of different permeabilities to determining the lysosomal pH. Using these methods we find that isolated lysosomes carry a minimal potassium conductance. This is also consistent with data obtained this year monitoring acidification in isolated lysosomes under a range of ionic conditions. To tie these approaches together, we, in collaboration with Dr. Michael Grabe at the University of Pittsburgh, are computationally simulating the known features of the lysosomal acidfication mechanism to determine whether these can explain observed acidification behaviors, a manuscript published this year. In addition to our work on lysosomes, we have begun to explore the acidification process in other organelles. In particular, we have developed methods to isolate clathrin-coated vesicles from bovine brain. These vesicles are known to acidify using a v-type ATPase and have been suggested to use an anion as a counterion for acidification. This year we demonstrated that Cl- is is critical for v-ATPase induced acidficiation in our CCV prep and that monovalent cations have minimal effects. We are currently working to identify the specific vesicle population undergoing acidification in these preps and to identify the transporter mediating this counterion pathway.